Early detection of high-grade squamous intraepithelial
lesions in the cervix with quantitative spectroscopic
imaging
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Citation
Lau, Condon. “Early detection of high-grade squamous
intraepithelial lesions in the cervix with quantitative spectroscopic
imaging.” Journal of Biomedical Optics 18, no. 7 (July 1, 2013):
076013. © 2013 Society of Photo-Optical Instrumentation
Engineers
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http://dx.doi.org/10.1117/1.jbo.18.7.076013
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Early detection of high-grade squamous
intraepithelial lesions in the cervix with
quantitative spectroscopic imaging
Condon Lau
Jelena Mirkovic
Chung-Chieh Yu
Geoff P. O’Donoghue
Luis Galindo
Ramachandra Dasari
Antonio de las Morenas
Michael Feld
Elizabeth Stier
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Journal of Biomedical Optics 18(7), 076013 (July 2013)
Early detection of high-grade squamous intraepithelial
lesions in the cervix with quantitative spectroscopic
imaging
Condon Lau,a* Jelena Mirkovic,a† Chung-Chieh Yu,a‡ Geoff P. O’Donoghue,a§ Luis Galindo,a
Ramachandra Dasari,a Antonio de las Morenas,b Michael Feld,a∥ and Elizabeth Stierc
a
Massachusetts Institute of Technology, George R. Harrison Spectroscopy Laboratory, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139
Boston Medical Center, Department of Pathology, One Boston Medical Center Place, Boston, Massachusetts 02118
c
Boston Medical Center, Department of Obstetrics and Gynecology, One Boston Medical Center Place, Boston, Massachusetts 02118
b
Abstract. Quantitative spectroscopy has recently been extended from a contact-probe to wide-area spectroscopic
imaging to enable mapping of optical properties across a wide area of tissue. We train quantitative spectroscopic
imaging (QSI) to identify cervical high-grade squamous intraepithelial lesions (HSILs) in 34 subjects undergoing the
loop electrosurgical excision procedure (LEEP subjects). QSI’s performance is then prospectively evaluated on
the clinically suspicious biopsy sites from 47 subjects undergoing colposcopic-directed biopsy. The results
show the per-subject normalized reduced scattering coefficient at 700 nm (An ) and the total hemoglobin concentration are significantly different (p < 0.05) between HSIL and non-HSIL sites in LEEP subjects. An alone retrospectively distinguishes HSIL from non-HSIL with 89% sensitivity and 83% specificity. It alone applied prospectively on
the biopsy sites distinguishes HSIL from non-HSIL with 81% sensitivity and 78% specificity. The findings of this
study agree with those of an earlier contact-probe study, validating the robustness of QSI, and specifically An , for
identifying HSIL. The performance of An suggests an easy to use and an inexpensive to manufacture monochromatic
instrument is capable of early cervical cancer detection, which could be used as a screening and diagnostic tool for
detecting cervical cancer in low resource countries. © 2013 Society of Photo-Optical Instrumentation Engineers (SPIE) [DOI: 10.1117/1
.JBO.18.7.076013]
Keywords: cervix; cancer; dysplasia; spectroscopy; imaging.
Paper 12521RR received Aug. 14, 2012; revised manuscript received May 31, 2013; accepted for publication Jun. 3, 2013; published
online Jul. 10, 2013.
1
Introduction
Early detection is a key to reducing cervical cancer-related mortality. Cervical cancer typically is preceded by preinvasive
changes known as high-grade squamous intraepithelial lesion
(HSIL). The American College of Obstetrics and Gynecologists
recommends that women with HSIL receive treatment of the
cervical transformation zone (Tzone), typically with the loop
electrosurgical excision procedure (LEEP), to prevent further
progression of the disease. Therefore, for effective cervical
cancer prevention, it is critical to identify the women with cervical HSIL.
The current clinical practice in cervical cancer screening
involves cervical cytology, i.e., a Papanicolaou test (Pap test)
for the population screening. If the Pap test result is abnormal,
a direct visual examination of the cervix under magnification
(colposcopy) is conducted to guide biopsy of suspicious
*Present affiliation: Hong Kong University of Science and Technology, Division of
Biomedical Engineering, Kowloon, Hong Kong SAR, China.
†
Present affiliation: Brigham and Women’s Hospital, Department of Pathology,
Boston, Massachusetts 02115.
‡
Present affiliation: Canon U.S.A., Inc., Optics Research Laboratory, 9030 South
Rita Road, Suite 302, Tucson, Arizona 85747.
§
Present affiliation: University of California, Department of Chemistry, Berkeley,
California 94709.
∥
Author is deceased.
Address all correspondence to: Condon Lau, Massachusetts Institute of
Technology, George R. Harrison Spectroscopy Laboratory, 77 Massachusetts
Avenue, Cambridge, Massachusetts 02139. Tel: (617) 2587667; Fax: (617)
2534513; E-mail: condonlau@ust.hk
Journal of Biomedical Optics
tissues, the histopathological examination of which serves as
the final diagnosis or gold standard. In the past decade, some
countries have added human papillomavirus testing as an additional cervical cancer screening test for women over the age 30
(Ref. 1); however, a patient with abnormal screening is still
referred for a colposcopic evaluation. Colposcopy and subsequent histopathological evaluation, even when conducted
by experts, is subject to significant diagnostic variability.2,3 A
technology that can reduce diagnostic variability by using a
more objective and ideally quantitative diagnostic scheme,
while maintaining the ability of colposcopy to examine the
entire cervix, would present a significant clinical advancement.
Furthermore, if this technology is inexpensive and easy to use, it
may be able to serve as a combined screening and diagnostic
tool for cervical cancer prevention in developing countries,
where the lack of medical infrastructure and funding have prevented implementation of a Pap test-based cancer prevention
program.
Wide-area spectroscopic imaging is an extension of contactprobe optical spectroscopy developed to quantitatively detect
HSIL and cancer in the cervix and other organs of the
human body. Research conducted in our laboratory, as well
as others, has demonstrated the ability of contact-probe
spectroscopy technologies to detect dysplasia in a variety of
organs including esophagus,4,5 skin,6 oral cavity,7,8 breast,9,10
and cervix.11–17 In general, contact-probe spectroscopy has
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demonstrated an ability to quantitatively distinguish dysplastic
and cancerous cervical tissue from normal tissue, and may be
more accurate than colposcopic diagnoses alone, which are
subjective.
In this paper, we conduct an in vivo study to extend quantitative spectroscopy from contact-probe to wide-area quantitative
spectroscopic imaging (QSI) for the detection of HSIL in the
cervix. Imaging offers an advantage over probes in which the
entire cervix can be examined in one measurement. This study
follows up on a contact-probe study done by our group,18,19 and
focuses on training and prospectively evaluating QSI’s ability to
distinguish HSIL from non-HSIL sites on the cervical Tzone.
Since the vast majority of HSILs are found in the Tzone,20
focusing on identifying HSIL within the Tzone reduces the
impact of normal anatomical variations on spectroscopy measurements and the likelihood of reporting high performance
metrics for clinically less important comparisons (e.g., including
significant numbers of normal ectocervix sites in the non-HSIL
group).11,18,21 We present the spectroscopic parameters observed
with QSI on subjects who underwent LEEP (LEEP subjects) and
women who underwent colposcopy with direct biopsy (colposcopy subjects). We normalize spectroscopy parameters to each
subject individually and train QSI to distinguish HSIL from nonHSIL in the LEEP data set. The spectroscopic criteria are then
applied prospectively to the colposcopy data set.
2
2.1
Materials and Methods
Instrumentation
We had previously developed a QSI system for early cancer
detection with reflectance and fluorescence spectroscopy capabilities.22 For this study, the QSI system was modified to have
the functions of a digital colposcope including acquiring whitelight photographs of the cervix with additional spectroscopic
capabilities. In brief, colposcopic QSI illuminated a 1-mm diameter region of the cervix with visible and 337-nm light, and collected reflectance and fluorescence spectra returning from a
concentric 2-mm diameter region over the visible wavelengths
(400 to 700 nm). The measured spectra were fit to light propagation and fluorescence models to extract the amounts of native
scatterers, absorbers, and fluorophores present.23,24 Once the
measurement was complete for one region, raster scanning
was used to interrogate another 1-mm diameter region until a
2.1 × 2.1 cm2 area of the cervix was examined. The total acquisition time was approximately 80 s. After the spectra were
processed (offline), we obtained maps of six spectroscopy
parameter: A (reduced scattering coefficient in units of mm−1
at 700 nm), B (wavelength dependence of reduced scattering
coefficient), [Hb] (total hemoglobin concentration in units of
mg∕mL), α (oxygen saturation), Coll (collagen concentration
in arbitrary units), and NADH (concentration of the reduced
form of nicotinamide adenine dinucleotide in arbitrary units).
These 21 × 21 pixel maps showed the value of a spectroscopy
parameter at multiple points on the cervix. The QSI system compensated for the motion during the acquisition time using the
white-light photographs as previously described.22 Note that
this study and earlier studies had not documented the concentrations of scatterers, absorbers, and fluorophores in vivo with
other methods. The quantitative in QSI referred to calibration
with tissue phantoms of known optical properties.
Journal of Biomedical Optics
2.2
Clinical Data Acquisition
The QSI system was used to examine subjects undergoing colposcopic evaluation at the Boston Medical Center (BMC). All
aspects of the study were approved by the Institutional Review
Boards of the Massachusetts Institute of Technology and the
BMC. Women who were pregnant, under the age of 18 or
over 65, or non-English speaking were excluded from the
study. Study participation was offered to all eligible subjects,
and written informed consent was obtained from study participants. Five percent acetic acid was applied to the cervix to
remove residual mucous and to initiate the acetowhitening
effect. The QSI system was positioned at the correct orientation
and distance (22.5 cm) from the subject’s cervix, and data was
collected. Data collection started approximately 5 min after
acetic acid application. After the QSI scan, the subject underwent her scheduled procedure. Thirty-four subjects undergoing
colposcopic evaluation prior to their scheduled LEEP and
47 women undergoing colpsocopy with directed biopsy were
recruited for the study. The 34 LEEP subjects were referred
based on a prior colposcopy and subsequent HSIL histopathology diagnosis. For the colposcopy subjects, only clinically suspicious sites were biopsied; the majority of the biopsies were
from the Tzone.
2.3
Histopathological Evaluation (LEEP)
Upon removal from the subject, the 12, 3, 6, and 9 o’clock positions on the squamo-columnar junction (SCJ) of the LEEP
specimen were marked with color pins. Specimens were submitted to the pathology department at BMC and sectioned, stained,
and evaluated per standard of care, which included sectioning
the specimen into 12 pieces corresponding to the hour hands
of a clock. This, combined with the four pins and the whitelight photographs acquired by the QSI system, was used to
correlate pathology with specific pixels on the spectroscopy
parameter maps. The lead pathologist on the study (Antonio
de las Morenas) classified every millimeter of all 12 sections
as ectocervix, HSIL Tzone, non-HSIL Tzone, endocervix, or
denuded mucosa. Note that HSIL can be further classified as
cervical intraepithelial neoplasia (CIN) 2 or 3 (Ref. 25). NonHSIL included low-grade squamous intraepithelial lesions
(LSILs) and nondysplastic tissue. No invasive cancer was
observed in this study. Endocervical lesions, such as adenocarcinoma, were not evaluated in this study. The position of each
millimeter along the section was measured from the SCJ, which
was clearly visible on the slides and in the photographs. In sections where the SCJ was not visible in both the slides and the
photographs, no position reference was recorded. For each subject, the slide(s) with the most severe disease, as identified by the
lead pathologist, were also examined by two other pathologists
(Christopher Crum and Sandra Cerda). They independently
determined the most severe disease present on the subject
(HSIL or non-HSIL). The consensus diagnosis of the most
severe disease was formed from that of the majority. Only measurements from specimens where the lead pathologist’s diagnosis of the most severe disease agreed with that of the consensus
were used to train and evaluate QSI’s ability to detect HSIL.
However, measurements from all LEEP subjects were used to
help determine endocervix and ectocervix (discussed below).
The end result of the LEEP pathology analysis was a disease
map showing the locations of HSIL on the subject (refer
to Fig. 1).
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Fig. 1 High-grade squamous intraepithelial lesion (HSIL) and non-HSIL
(low-grade SIL and benign) regions overlaid on a white-light photograph
of the cervix. The disease state and locations were determined by loop
electrosurgical excision procedure (LEEP) pathology, where the excised
tissue is cut into 12 slices corresponding to the hour positions on a
clock, and examined histopathologically. Solid white lines indicate
regions of the cervix confirmed to have HSIL by pathology. HSIL
may be present in other parts of the cervix, but these sites could not
be confirmed due to denuded mucosal surfaces or finite pathology
sampling. Dashed white lines indicate non-HSIL regions confirmed
by pathology. Green lines indicate the squamo-columnar junction
(SCJ), the boundary between endocervix and transformation zone
(Tzone). No diagnosis was available at the LEEP specimen positions
10 and 11 o’clock due to denuded mucosa and part of an intrauterine
device is visible.
2.4
Histopathological Evaluation (Colposcopy with
Directed Biopsy)
Each biopsy specimen was submitted to pathology, where it was
evaluated by three pathologists who independently rendered
diagnoses of HSIL or non-HSIL. A consensus diagnosis was
formed from the majority decision, and this served as the
gold standard. Pathology’s diagnosis for each biopsy was compared with QSI’s diagnosis by using the photograph with the
location of the biopsy indicated to determine the pixel on the
diagnostic map closest to the biopsy site. In this fashion, we
evaluated the accuracy of QSI, used as an adjunct to colposcopy,
by providing a diagnosis of HSIL or non-HSIL at clinically suspicious sites.
2.5
Data Analysis (LEEP)
Spectroscopy parameters, measured from HSIL and non-HSIL
pixels in the Tzones of LEEP subjects, were selected by correlating the disease maps to the spectroscopy parameter maps.
This was accomplished by identifying the 12, 3, 6, and 9 o’clock
positions on the SCJ of each subject using the white-light photographs acquired by the QSI system.22 Since the photographs
were acquired simultaneously with the parameter maps, each
millimeter on the disease map was matched with the pixels
of the parameter maps (refer to Fig. 1). We did not include
the effects of possible specimen shrinkage following fixation.26
Pixels corresponding to denuded mucosa or sections without a
visible SCJ were not included in the subsequent analyses.
Spectroscopy parameters measured from ectocervix pixels
on all subjects were used for normalizing spectroscopy parameter maps. There are normal inter-subject variations in tissue
Journal of Biomedical Optics
properties due to factors such as age and menstrual
cycle.14,27–29 These are reflected in spectroscopy parameters,
and may confound contrast due to disease.28,30 We normalized
all spectroscopy parameter maps per subject to counteract the
impact of normal variations. Normalization for each parameter
was done by dividing the value at each pixel of the parameter
map by the average value of the parameter measured from pixels
corresponding to normal ectocervix. Normalized parameters
were denoted by a subscript n (A versus An ). A similar normalization procedure was applied in our recent contact-probe study,
and yielded improved distinction of HSIL from non-HSIL.19
To train QSI to identify ectocervix pixels for normalization,
the A and Coll values from colposcopically defined ectocervix
and nonectocervix pixels were used to form the likelihood functions in a Bayesian analysis. Schomacker et al. previously
observed that normal squamous can be distinguished from
CIN 2 and 3 using fluorescence spectra.31 We used the number
of ectocervix pixels and the total number of cervix pixels (total
number of pixels covering the cervices of the LEEP subject
including ectocervix pixels) to form the prior probability. The
Bayesian analysis was applied to the spectroscopy data from
all LEEP subjects to identify ectocervix pixels given the
local A and Coll values (posterior probability greater than
0.5). For each subject, all such ectocervix pixels surrounded
by at least eight other such ectocervix pixels (continuity criterion) were used as the spectroscopy parameter normalization
region. Normalized spectroscopy parameter maps of each subject were formed by dividing each regular parameter map by the
average value of the corresponding parameter in ectocervix
pixels of the subject. Note that pixels not chosen for normalization may span the endocervix, Tzone, and ectocervix.
Choosing pixels for normalization does not imply the remaining
pixels define the Tzone.
2.6
Data Analysis (Colposcopy)
The Bayesian analysis and continuity criterion were also applied
to the spectroscopy data from all colposcopy subjects to identify
ectocervix pixels. The likelihood functions and prior probability
were obtained from the LEEP data. Normalized spectroscopy
parameter maps of each subject were formed by dividing
each regular parameter map by the average value of the corresponding parameter in ectocervix pixels of the subject.
3
Results
A total of 34 LEEP subjects and 47 colposcopy subjects were
recruited for this study. The study participants consisted of 43
self-identified blacks, 19 whites, 16 Hispanic/Latinos, 2 Asians,
and 1 other. Their ages had a mean of 31.3 years and a standard
deviation of 9.7 years. Twenty of the subjects had referral cytology of atypical squamous cells of undetermined significance
(ASCUS), three had atypical squamous cells cannot rule out
a high-grade lesion (ASC-H), 36 had LSIL, 18 had HSIL,
and four had other. Among the LEEP subjects, two had referral
cytology negative for intraepithelial lesion or malignancy, seven
had ASCUS, one had ASC-H, 12 had LSIL, 10 had HSIL, and
two had unknown. Since this study is the initial clinical deployment of the QSI system, the first four LEEP subjects were
required to optimize the equipment and to repair problems.
Spectroscopic results from the remaining 30 LEEP subjects
are presented. The lead pathologist’s diagnosis agreed with
that of the consensus in 25 of the LEEP subjects, and these
are used to train QSI to identify HSIL. All 30 LEEP subjects
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Table 1 The number of subjects undergoing different numbers of colposcopically directed biopsies. All biopsies were performed due to
clinical need only. Other refers to subjects without spectroscopy
data due to instrument problems, and their pathology results were
not recorded in the study. Note that there was no spectroscopy data
at 10 of the biopsy sites due to blood, specular reflection, or the site
not being covered by the scan. No pathology data was available at
four of the biopsy sites due to specimen damage. This resulted in
the 67 biopsy sites with spectroscopy data in the study.
# of biopsies
0
1
2
3
4
Other
# of subjects
5
16
10
11
3
2
Table 2 The number of subjects undergoing different numbers of
colposcopically directed biopsies that were histopathologically diagnosed as high-grade squamous intraepithelial lesion (HSIL). No spectroscopy data was available at two of the HSIL-biopsy sites. This
resulted in the 16 HSIL-biopsy sites with spectroscopy data in the study.
# of HSIL biopsies
0
1
2
3
4
Other
# of subjects
34
6
4
0
1
2
with spectroscopy measurements were used to identify ectocervix. Eighteen of the 25 LEEP subjects had histopathologically
diagnosed HSIL. The colposcopy study yielded 16 HSIL and 51
non-HSIL biopsies. Table 1 shows the number of subjects
undergoing different numbers of colposcopically directed biopsies. Table 2 shows the number of subjects undergoing different
numbers of colposcopically directed biopsies which were
histopathologically diagnosed as HSIL. HSIL biopsies were
obtained from 11 of the colposcopy subjects.
QSI measures tissue spectroscopic properties at multiple sites
across the cervix. Figure 2 shows all six spectroscopy parameter
maps measured from a representative cervix. The dark slit near
the center is the cervical os, the opening of the endocervical
canal. Away from the os near the edge of the cervix is the ectocervix, which is characterized by higher scattering [upper and
lower portions of Fig. 2(a)]. Between the endocervix and
ectocervix, spectroscopy parameters transition from columnar
to squamous values. The region between the endocervix and
ectocervix is the Tzone. In general, the Tzone has lower A,
lower B, higher [Hb], lower α, lower Coll, and higher
NADH than ectocervix.
Spectroscopy parameters in this study are normalized by
their respective average values across ectocervix pixels of the
same subject. Figure 3 shows the six box plots of normalized
parameters measured from all HSIL and non-HSIL pixels of
the LEEP subjects. HSIL pixels exhibit considerably lower
scattering (An ) than non-HSIL pixels [Fig. 3(a)]. The p-value
of this difference, computed using the two-tailed t-test, is 0
(beyond computer precision). HSIL also has slightly lower Bn
[p > 0.05, Fig. 3(b)], αn [p > 0.05, Fig. 3(d)], and Colln
[p > 0.05, Fig. 3(e)], and slightly higher ½Hbn [p < 0.01,
Fig. 3(c)] and NADHn [p > 0.05, Fig. 3(f)] than non-HSIL.
However, these differences are small when compared with
the An difference. Applying a threshold of An < 0.83 (HSIL)
and An > 0.83 (non-HSIL) on the boxplot of Fig. 3(a), HSIL
can be distinguished from non-HSIL with 89% sensitivity
and 83% specificity. Note that these two values are reported
for all HSIL and non-HSIL pixels. The 0.83 An threshold maximizes the sum of sensitivity and specificity in the LEEP data
set. Therefore, QSI can retrospectively distinguish HSIL from
Fig. 2 Spectroscopy parameter maps acquired by quantitative spectroscopic imaging (QSI) overlaid on a white-light photograph of a cervix. (a) A,
reduced scattering coefficient at 700 nm (mm−1 ). (b) B, wavelength dependence of reduced scattering coefficient. (c) [Hb], total hemoglobin concentration (mg∕mL). (d) α, oxygen saturation (%). (e) Coll, collagen concentration (a.u.). (f) NADH, concentration of reduced form of nicotinamide
adenine dinucleotide (a.u.). Pixels with no spectroscopy data due to the presence of specular reflection, motion, acute tissue angle relative to QSI
system, or noncervical substances are uncolored in the parameter maps. Note that specular reflection observed in the white-light photographs is not
necessarily sites of specular reflection for spectroscopy measurements. A portion of the speculum (bottom of photograph) is visible.
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Fig. 3 Box plots of normalized spectroscopy parameters measured from HSIL and non-HSIL pixels in the excised region of the LEEP subjects.
Normalization was performed by dividing spectroscopy parameters by their respective average values across ectocervix pixels of the same subject.
(a) An , normalized A. (b) Bn , normalized B. (c) ½Hbn , normalized [Hb]. (d) αn , normalized α. (e) Colln , normalized Coll. (f) NADHn , normalized NADH.
The horizontal center line is the median and the lower and upper edges of the rectangular box indicate the 25th and 75th percentiles, respectively. The
horizontal lines at the ends of the dashed vertical line indicate the extent of the data, and the crosses indicate outliers.
Fig. 4 Receiver operator characteristic (ROC) curve (solid line) for
distinguishing HSIL from non-HSIL pixels in the LEEP subjects. Only
An was used in the analysis. The dashed line indicates the line of
chance.
non-HSIL in LEEP subjects with 89% sensitivity and 83%
specificity, which is in good agreement with an earlier contact-probe study.19
Figure 4 shows the entire receiver operator characteristic
(ROC) curve for distinguishing HSIL from non-HSIL in the
LEEP subjects computed using An only. The ROC curve
shows different retrospective sensitivities and specificities that
can be achieved with different An thresholds (the An ¼ 0.83
threshold is included in the ROC curve). The area under the
ROC curve, a measure of QSI’s ability to distinguish HSIL
from non-HSIL, is 0.93 (1.0 is perfect distinction, and 0.5 is
equivalent to chance).
Figure 5 shows the mean reflectance and fluorescence spectra acquired from clinically suspicious biopsy sites of colposcopy subjects. The differences between HSIL and non-HSIL
spectra are small. Note that these spectra are not “normalized”
like the spectroscopy parameter maps. QSI with an An threshold
of 0.83 was then applied prospectively to the biopsy sites to distinguish HSIL from non-HSIL. Note that both the An threshold
and the A and Coll values for identifying ectocervix were chosen
from the LEEP data only. Table 3 shows the 13 true positives, 11
false positives, 40 true negatives, and 3 false negatives. QSI can
prospectively distinguish HSIL from non-HSIL in colposcopy
Journal of Biomedical Optics
Fig. 5 Mean reflectance (a) and fluorescence (b) spectra acquired from
the clinically suspicious biopsy sites of colposcopy subjects. Solid lines
indicate non-HSIL sites, and dashed lines indicate HSIL sites. Note that
these spectra are not “normalized” like the spectroscopy parameter maps.
subjects with 81% sensitivity and 78% specificity. This is a measure of QSI’s performance identifying HSIL when used as an
adjunct to colposcopy. Combining colposcopy with spectroscopy would require 13 (true positives) þ11 (false positives)
biopsies be taken from the 47 colposcopy subjects. This
comes to 0.51 biopsies per subject.
Table 3 Prospective evaluation of quantitative spectroscopic imaging’s (QSI’s) ability to distinguish HSIL from non-HSIL in the biopsy
sites of colposcopy subjects. The rows are QSI’s diagnoses, and the
columns are consensus pathology’s classifications. There are 13 true
positives, 11 false positives, 3 false negatives, and 40 true negatives.
HSIL QSI (An < 0.83)
Non-HSIL QSI (An > 0.83)
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HSIL biopsy
Non-HSIL biopsy
13
11
3
40
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4
Discussion
The results presented in this paper demonstrate QSI’s ability to
measure cervical tissue properties in vivo. This measurement of
reflectance and fluorescence properties is equivalent to several
hundred contact-probe measurements (Fig. 2), and can be completed in a relatively short period of time. Spectroscopy parameter maps measured from the cervix show that the Tzone has
lower A, lower B, higher [Hb], lower α, lower Coll, and higher
NADH than ectocervix. We also find HSIL pixels exhibit considerably lower normalized A than non-HSIL pixels in the LEEP
subjects, while other normalized parameters exhibit relatively
little difference between the disease states. QSI can retrospectively distinguish HSIL from non-HSIL in the LEEP
subjects with 89% sensitivity, 83% specificity, and area under
ROC curve ¼ 0.93 using An alone. Using the thresholds
obtained from the LEEP data set, QSI can prospectively distinguish HSIL from non-HSIL in colposcopy subjects with 81%
sensitivity and 78% specificity.
Previous efforts have been made to combine spectroscopy
with wide-area imaging in the cervix32–43 to enable wide-area
surveillance as is done in colposcopy. For example,
Milbourne et al.32 and Park et al.36 conducted three color reflectance and fluorescence spectroscopic imagings, and observed
that dysplastic tissue emits slightly different colors than normal
tissue when excited with ultraviolet or blue excitation light.
Alvarez et al. conducted a randomized trial using the LUMA
cervical imaging system to determine if combining reflectance
and fluorescence spectroscopic imaging with colposcopy on
subjects referred for colposcopy would increase HSIL detection.33 They determined that combining spectroscopy with
colposcopy improved the true positive rate of HSIL detection
among subjects with ASC or LSIL cytology by 26% over
colposcopy alone. However, this benefit came at the expense
of a 33% increase in the number of biopsies taken from those
subjects. Huh et al. similarly observed that spectroscopyþ
colposcopy increased the detection of HSIL by 25% among
women with ASC/LSIL cytology.43
QSI differs from previous spectroscopic imaging techniques
in two significant ways. First, QSI is the imaging implementation of quantitative spectroscopy, which means it measures tissue properties by using biophysical models of light propagation
to extract information about tissue scatterers, absorbers, and fluorphores from the measured spectra.22–24 Modeling allows QSI
to directly measure tissue structural and biochemical changes
associated with disease. Without modeling, spectral measurements may vary significantly with instrument configuration.44
Several contact-probe spectroscopy studies have also used
biophysical modeling to measure tissue changes.11,13,21,45,46
Understanding these changes will allow knowledge obtained
from QSI studies done with one instrument implementation
to be transferred to another implementation, and potentially
facilitates extending QSI to detect early cancer in other body
organs. Second, QSI uses narrow-field illumination, while
other systems use wide-field illumination. Narrow-field illumination results in a smaller sampling volume at each pixel, similar
to the difference between confocal and bright field microscopy,
and thus, likely allows QSI to detect smaller dysplasia.
In addition to biophysical modeling, our proposed QSI diagnostic approach specifically helps to address the effects of intersubject variability. Normalized spectroscopy parameters that
highlight contrast within the same subject are used, because
diagnosis with the regular parameters can couple intra- and
Journal of Biomedical Optics
inter-subject contrast. Inter-subject contrast may be due to normal cervical variations, such as age and menopausal status, or
time after acetic acid application.11,14,28,29,47 Normalization was
also used successfully by Mirkovic et al. to distinguish HSIL
from non-HSIL with a contact-probe implementation of quantitative spectroscopy.19 In that study, the An parameter was
obtained by dividing spectroscopy parameters measured at
clinically suspicious sites (biopsied after measurement) by
the respective parameters measured at normal ectocervix sites.
The authors reported retrospective sensitivity and specificity of
89% and 79%, respectively, distinguishing HSIL from nonHSIL at the biopsy sites of colposcopy subjects.
This study focuses on distinguishing HSIL from non-HSIL
in the cervical Tzone, which reduces the impact of normal anatomical variations on spectroscopy parameters and also the likelihood of reporting high performance metrics for clinically less
important comparisons (e.g., distinguishing HSIL from normal
ectocervix).18 The changes in An observed with HSIL are likely
due to QSI measuring changes in stromal density that occur due
to degradation by matrix metalloproteinases.48 This can reduce
the amount of scattering from the stroma, which has significant
influence on tissue reflectance spectra.49 Several earlier studies
have observed a similar trend in their data. Nordstrom et al.
reported that reflectance spectroscopy alone helped to distinguish HSIL from nondysplastic Tzone, and fluorescence measurements were not effective.50 Similarly, Mirabal et al. noted a
decrease in tissue reflectivity with the severity of dysplasia.51
However, because these authors did not conduct quantitative
spectroscopy, they could not separate the scattering and absorption contributions to reflectance, as we did in this paper, which
allows us to observe the importance of scattering measurements alone.
Acetic acid was applied to the cervix in this study prior
to QSI. The subsequent aceto-whitening changes would be
expected to increase scattering in HSIL relative to non-HSIL.
However, this study observed lower scattering (i.e., An ) in
HSIL. There are several potential reasons for the discrepancy.
In this study, QSI started approximately 5 min after acetic acid
application. The most optically apparent and dynamic acetowhitening changes occur within five minutes of application.38,40
The remaining aceto-whitening changes likely affect reflectance
at short wavelengths more than at the long wavelengths, where An
is measured. Wu and Qu observed that backscattering by cell
monolayers increased after acetic acid application, more so at
short than at long wavelengths.52 In vivo, increased backscattering
may decrease the amount of short wavelength light penetrating
the epithelium and reaching the blood vessels, thus increasing
reflectance. This effect is less pronounced at long wavelengths,
where An is measured, because hemoglobin absorption is minimal. Due to the above reasons, aceto-whitening is unlikely to be
contradictory to the results of this study.
This study prospectively evaluates QSI’s ability to distinguish HSIL from non-HSIL at the clinically suspicious biopsy
sites of colposcopy subjects. One of the potential applications of
this research is an optical system that is used with standard colposcopy to identify biopsy sites. Compared with colposcopy
alone, such a combined system may reduce the number of biopsies required to sample the cervix without considerably reducing
HSIL detection. Future studies will be performed to determine
the number of biopsies called by the combined system, the number of HSIL sites detected, and the number of subjects with
HSIL detected. A HSIL pathology result was chosen as the
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Lau et al.: Early detection of high-grade squamous intraepithelial lesions in the cervix. . .
threshold in this study, because it is the current threshold for
treatment (e.g., by LEEP). An alternative threshold is a CIN
3 pathology result. In this study, CIN 2 and 3 pathology results
were grouped together as HSIL pathology. However, if the system were tuned to identify only CIN 3 pathology, there would
be a significant likelihood of missing biologic CIN 3. This is
because a CIN 2 pathology result is often misclassified as biologic CIN 3 (Ref. 53). Therefore, HSIL pathology is the more
suitable threshold for this QSI study.
The QSI system described so far in this study is not suitable
for use in a low-resource setting. However, the finding that
measuring the reduced scattering coefficient at one wavelength
can distinguish HSIL from non-HSIL permits the design of a
significantly simpler, faster, and less-expensive optical system
for cervical dysplasia detection. The simple optical system’s
simplicity may facilitate its use as a screening and diagnostic
tool in low-resource developing countries, which lack the medical infrastructure for implementing cervical cancer screening
programs. This system, potentially based on a red laser pointer
and an optical power meter, could cost very little to manufacture
and would be easy to use. The system can likely be operated
with battery power, and will not require computer control. A
properly designed container should shield the optics from
water, dust, and dirt. The simple optical system will also be
a valuable addition to a standard colposcope in the developed
countries.
The operator of the simple optical system will be trained to
identify ectocervix sites for normalization. Such sites are typically located far from the SCJ and Tzone. Study is required to
determine visual features for identifying normalization sites,
although we expect the features to be similar to those of ectocervix as viewed through a magnifying glass. Identifying normalization sites should require less training than performing
standard colposcopy. Also note that the simple system will
likely be employed in low-resource settings without a pathology
lab. Therefore, any subject with a low An measurement would be
treated on the spot without taking biopsies. Whether or not this
leads to an excessive number of unnecessary treatments will be
investigated. Sampling strategies will be devised to optimize the
balance between sensitivity and specificity.
Acknowledgments
All authors are supported by National Institutes of Health Grants
R01 CA097966 and P41 RR02594 (Laser Biomedical Research
Center). The authors thank Dr. Kamran Badizadegan for his
contributions to the study design and data interpretation,
Victoria Feng and Cecilia Marquez for their contributions
to the data acquisition, and Dr. Sandra Cerda and
Dr. Christopher Crum for their contributions to the histopathological evaluation.
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